Background Preterm neonates are repeatedly exposed to many stressors in initial days of life such as environmental stressor (light and noise) and caregiver interventions (weighing, oral and tracheal suctioning, drawing blood samples, etc.). If preterm neonates’ stress is not recognized and left untreated, there may be long-term effects. Objective To assess the neonatal stress behavior among preterm neonates admitted in neonatal intensive care unit (NICU), Nehru Hospital, PGIMER, Chandigarh. Methodology A descriptive study was conducted to assess neonatal stress behavior among preterm neonates admitted in NICU, Nehru Hospital, PGIMER, Chandigarh. Ethical clearance was taken from Institutional Ethics Committee of PGIMER, Chandigarh. Total 100 participants admitted in NICU during the period of July–December 2022 were enrolled for the study using consecutive sampling technique. Data was collected by interviewing mother using interview schedule. It comprised of sociodemographic cum clinical profile of the preterm neonate. Observation was made by using—(a) modified infant positioning assessment tool (IPAT), (b) neonatal stress scale, and (c) preterm neonate’s behavior assessment scale. Results The present study shows that 88% of the preterm neonates show moderate stress and 70% of preterm neonates exhibit suspected abnormal behavioral response in terms of autonomic visceral subsystems, while 54% of the preterm neonates show suspected abnormal behavioral response in terms of state regulation attention interaction subsystem. Conclusion The present study concluded that moderate stress level and suspected abnormal behavioral response were found among hospitalized preterm neonates.